Stability of zopiclone in whole blood
‐ Studies from a forensic perspective
Gunnel Nilsson Division of Drug Research Department of Medical and Health Sciences Linköping University, Sweden Linköping 2010Supervisors Robert Kronstrand, Associate Professor Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Sweden Johan Ahlner, Professor Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Sweden Fredrik C. Kugelberg, Associate Professor Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Sweden Gunnel Nilsson, 2010 Published article has been reprinted with permission of the copyright holder. Paper I © 2010 Elsevier, Forensic Science International Printed in Sweden by LiU‐Tryck, Linköping, Sweden, 2010 ISBN 978‐91‐7393‐339‐1 ISSN 1100‐6013
Dala‐Gård Ring the bells that still can ring Forget your perfect offering There is a crack in everything That’s how the light gets in Anthem by Leonard Cohen
CONTENTS
ABSTRACT ... 1 POPULÄRVETENSKAPLIG SAMMANFATTNING... 3 LIST OF PAPERS ... 5 ABBREVIATIONS ... 6 INTRODUCTION ... 7 Pre‐analytical conditions... 7 Drug stability... 7 Design and evaluation of stability experiments... 9 Stability investigations of drugs... 10 Zopiclone... 11 Pharmacokinetics ... 12 Pharmacodynamics... 13 Forensic cases... 14 Analytical methods ... 16 Biological specimens... 17 AIMS OF THESIS ... 19 Specific aims ... 19 Paper I ... 19 Paper II... 19 MATERIALS AND METHODS ... 21 Study designs ... 21 Long‐ and short‐term stability ... 21 Freeze‐thaw stability... 22 Processed stability... 22 Degradation ... 22 Influence of pre‐analytical conditions... 22Ethical considerations ... 23 Equipment... 23 Chemicals and solutions ... 24 Analytical methods... 24 Gas chromatography ... 24 Liquid chromatography ... 26 Quality controls ... 26 Clinical chemical analysis ... 27 Statistical analysis ... 27 RESULTS... 29 Paper I ... 29 Long‐ term and short‐term stability ... 29 Freeze‐thaw stability... 31 Processed stability... 32 Degradation products... 32 Quality control samples ... 32 Paper II... 33 Authentic and spiked stability samples... 33 Quality control samples ... 35 GENERAL DISCUSSION... 37 Stability investigations... 37 Pre‐analytical aspects ... 40 Methodological aspects ... 40 CONCLUDING REMARKS... 43 Paper I ... 43 Paper II... 43 FUTURE PERSPECTIVES ... 45 ACKNOWLEDGEMENTS ... 47 REFERENCES... 49 APPENDIX (PAPERS I–II)... 59
ABSTRACT
Bio‐analytical results are influenced by in vivo factors like genetic, pharmacological and physiological conditions and in vitro factors like specimen composition, sample additives and storage conditions. The knowledge of stability of a drug and its major metabolites in biological matrices is very important in forensic cases for the interpretation of analytical results. Many drugs are unstable and undergo degradation during storage.
Zopiclone is a short‐acting hypnotic drug, introduced as a treatment for insomnia in the 1980s. However, this drug is also subject to abuse and can be found in samples from drug‐impaired drivers, recreational drug users and forensic autopsy cases. Zopiclone is analyzed in biological materials using different analytical methods. It is unstable in certain solvents and depending on storage conditions unstable in biological fluids. The aim of this thesis was to investigate the stability of zopiclone in human whole blood and to compare stability between authentic and spiked samples. Interpretation of zopiclone concentrations in whole blood is important in forensic toxicology. The following investigations were performed to study the stability of zopiclone in both spiked and authentic human blood.
First, different stability tests were performed. Spiked blood samples were stored at –20°C, 5°C and 20°C and the degradation of zopiclone was investigated in long‐ and short‐term stability. Authentic and spiked blood samples were stored at 5°C and differences in zopiclone stability were studied. Processed sample stability and effect of freeze/thaw cycles were also evaluated.
Second, influence of pre‐analytical conditions on the interpretation of zopiclone concentrations in whole blood was investigated. Nine volunteers participated in the study. Whole blood was obtained before and after oral administration of 2 x 5 mg Imovane®. Aliquots of authentic and spiked blood
were stored under different conditions and zopiclone stability was evaluated. In this study, the influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized.
Analyses of zopiclone were performed by gas chromatography with nitrogen phosphorous detection and zopiclone concentrations were measured at selected time intervals. Degradation product of zopiclone was identified using liquid chromatography‐tandem mass spectrometry.
The first study showed that zopiclone degrades in human blood depending on time and temperature and may not be detected after long‐term storage. The degradation product 2‐amino‐5‐chloropyridine was identified following zopiclone degradation. The best storage condition was at –20°C even for short storage times, because freeze‐thaw had no influence on the results. In butyl acetate extracts, zopiclone was stable for at least two days when kept in the autosampler. However, in blood samples stored at 20°C a rapid decrease in concentration, was noticed. This rapid degradation at ambient temperature can cause an underestimation of the true concentration and consequently flaw the interpretation.
The second study showed no stability differences between authentic and spiked blood but confirmed the poor stability in whole blood at ambient temperature. The results showed that zopiclone was stable for less than 1 day at 20°C, less than 2 weeks at 5°C, but stable for 3 months at –20°C. This study, demonstrates the importance of controlling pre‐analytical conditions from sampling to analysis to avoid misinterpretation of toxicological results.
POPULÄRVETENSKAPLIG
SAMMANFATTNING
Inom forensisk toxikologi undersöks förekomst av droger, läkemedel och gifter i biologiskt material. Resultatet av undersökningarna bidrar till bedömningar i rättsliga utredningar av drogmissbruk, drogpåverkan och dödsorsak. Kunskap om stabiliteten hos kemiska föreningar i biologiska prover under förvaring är av väsentlig betydelse både analytiskt och tolkningsmässigt. Många substanser är instabila och förändras under förvaring. Provmaterial transporteras via post, registreras på laboratoriet och förvaras därefter i kyl. Innan samtliga undersökningar är klara har provet normalt förvarats i en till två veckor. Rättsliga processer kan pågå under en längre tid och det händer ibland att prover måste undersökas på nytt när nya frågeställningar tillkommer. Provmaterialet kan då ha förvarats i flera veckor eller månader.
Zopiklon introducerades som läkemedel på 1980‐talet för behandling av kortvariga sömnbesvär. I Sverige finns zopiklon som den verksamma substansen i sömnmedlet Imovane®. Inom forensisk toxikologi undersöks
förekomst av zopiklon när analys av läkemedlet begärs. Zopiklon återfinns i såväl missbruksärenden, drograttfylleriärenden som i obduktionsfall. Zopiklon kan analyseras i olika biologiska material som till exempel helblod, urin, hår och postmortalt blod. Beroende på förvaringsförhållanden, materialets beskaffenhet och pH förändras mängden zopiklon i lösningar och i biologiskt material. Syftet med studierna i denna avhandling var att undersöka stabiliteten för zopiklon i helblod, samt att studera stabilitetsskillnader mellan blod innehållande zopiklon efter tillsats (spikade prover), med blod innehållande zopiklon efter intag av läkemedlet (autentiska prover). Kunskap om stabiliteten för zopiklon i detta material är viktig, eftersom resultat från analyser på helblod utvärderas och tolkas inom forensisk toxikologi.
Två olika studier genomfördes. I den första studien gjordes olika typer av stabilitetstester. Spikade prover förvarades vid −20°C, 5°C och 20°C och koncentrationerna av zopiklon följdes över tid. Stabilitet i provextrakt under förvaring på analysinstrument och stabilitet efter det att prov frysts och tinats undersöktes också.
I den andra studien undersöktes hur provhantering innan analys kan inverka på koncentrationerna av zopiklon i helblod och påverka tolkningen av resultat. I studien deltog nio frivilliga individer. Blodprov togs före och efter intag av 2 x 5 mg av läkemedlet Imovane®. Spikade och autentiska prover
förvarades vid −20°C, 5°C och 20°C och koncentrationerna av zopiklon följdes över tid och jämfördes. I denna studie kontrollerades även faktorer som indirekt kan ha en påverkan på substansens koncentration i helblod. Faktorer som materialets beskaffenhet, förekomst av andra droger och mängden av plasmaproteiner kontrollerades.
Den första studien visade att koncentrationerna av zopiklon i helblod sjunker under förvaring beroende på temperatur och tid. Vid provförvaring i rumstemperatur sjönk koncentrationen av zopiklon snabbt. Det har tidigare visats att när pH stiger förändras zopiklonmolekylen och degraderar via kemisk hydrolys till 2‐amino‐5‐klorpyridin. Denna degraderingsprodukt kunde identifieras vid ett enkelt försök på zopiklonspikade prover som inkuberats vid 37°C. Mängden aminoklorpyridin ökade i proportion till minskningen av mängden zopiklon. Mätning av degraderingsprodukten kan komma till nytta vid utredningar av förekomst av zopiklon i fall där provmaterial har förvarats under lång tid, till exempel vid speciella dödsfallsutredningar.
Frysning och tining av prov hade ingen inverkan på koncentrationerna av zopiklon i helblod. Zopiklon var stabilast vid förvaring i frys och eftersom frysning och tining inte påverkade analysresultatet, bör prover kunna förvaras i frys både under kortare och längre tidsperioder.
Zopiklon var stabilt under minst två dagar i provextrakt som förvarats i rumstemperatur på analysinstrumentet. Det innebär att om det uppstår oförutsedda problem under pågående analys, så är det möjligt att analysera provextraktet på nytt inom denna tidsperiod.
Den andra studien visade inga skillnader i stabilitet mellan spikade och autentiska prover och resultaten från stabilitetstesterna i denna studie bekräftade resultaten från den första studien. Zopiklon i helblod visade sig vara stabilt mindre än en dag vid förvaring i rumstemperatur, mindre än två veckor vid förvaring i kyl, men i minst tre månader vid förvaring i frys. Detta innebär att provmaterialets förvaring från provtagning fram till analys måste kontrolleras med avseende på temperaturförhållanden. Analysresultat från prover som förvarats en längre tid måste tolkas med stor försiktighet.
LIST OF PAPERS
This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I. Stability tests of zopiclone in whole blood. Nilsson GH, Kugelberg FC, Kronstrand R, Ahlner J. Forensic Sci Int. 2010, 200(1‐3):130‐135. II. Influence of pre‐analytical conditions on the interpretation of zopiclone concentrations in whole blood. Nilsson GH, Kugelberg FC, Ahlner J, Kronstrand R. Forensic Sci Int. 2010, accepted for publication.ABBREVIATIONS
CV Coefficient of variation CYP Cytochrome P450 GABA ‐aminobutyric acid GC Gas chromatography GHB Gamma‐hydroxybutyric acid HPLC High performance liquid chromatography LC Liquid chromatography LSD Lysergic acid diethylamide MS Mass spectrometry NPD Nitrogen‐phosphorus detector SEM Standard error of the mean SD Standard deviationTHCCOOglu 11‐nor‐Δ9‐carboxy‐tetra‐hydrocannabinolic glucuronide
THCCOOH 11‐nor‐Δ9‐carboxy‐tetra‐hydrocannabinolic acid
INTRODUCTION
Pre-analytical conditions
Laboratory activities are commonly classified in pre‐, intra‐ and post‐analytical processes. The pre‐analytical phase includes request, sample collection, transport, registration, preparation and aliquoting, storage, freezing and thawing [1]. The intra‐analytical phase covers the measurement procedures while the post‐analytical phase includes processing, verifying, interpreting and reporting of the results. In the past, the development of analytical technology and quality specifications has been the major focus. However, in clinical chemistry it was noticed that many problems occurred in the pre‐ analytical phase [2,3] and attention was directed to the pre‐analytical process in laboratory medicine as well as in forensic toxicology [4‐6].
Toxicological laboratory analysis results are influenced pre‐analytically by
in vivo factors like genetic, pharmacological and physiological conditions and in vitro factors like specimen composition, sample additives and storage
conditions. Pharmacokinetic and pharmacogenetic studies have shown that factors such as age, gender, ethnic origin, body weight, liver and kidney function, plasma/blood ratio and polymorphism of drug metabolizing enzymes as well as drug interactions must be considered when interpreting results [7‐10]. Analytical methods must be carefully validated for drug measurement and quantifications. Method validation includes several analytical parameters; selectivity, linearity, accuracy, precision, limit of quantification, limit of detection, recovery, robustness and nowadays even parameters affected by specimen composition such as matrix effects and stability [11‐13].
Drug stability
Stability has been defined as “The chemical stability of an analyte in a given matrix under specific conditions for given time intervals” [14]. In forensic toxicology, the analyte can be a drug, metabolite and/or a degradation product
in a biological matrix. Examples of biological matrices are whole blood, serum, plasma, urine, hair, oral fluids and tissues.
The knowledge of stability of a drug and its major metabolites in biological matrices is very important in forensic cases for the interpretation of analytical results [6,15,16]. In forensic investigations the pre‐analytical stability processes start at the time of sampling and proceeds until the time of analysis. Frequently, there is a delay of a few days between sampling, drug screening and drug quantification. In forensic toxicology supplementary analysis or reanalysis is sometimes necessary because of the legal process. In such cases it is not uncommon that samples are stored weeks or months before the final drug quantification is done. In post‐mortem forensic cases the storage of the body between the time of death and the time of sampling during the autopsy also has to be considered. A drug, which is present in a biological sample, may decompose during storage and may not be detected when the sample is analyzed.
The presence of drugs and poisons are tested in biological materials like blood, urine and hair [17,18]. The identification and the quantification of drug and metabolite concentrations in blood are valuable for the assessment of drug abuse in connection with crime and sometimes for establishing the cause of death. The time of sampling is important, especially if there is any suspicion of drug influence in the crime. Urine samples are useful in cases of drug misuse or abuse because the drug is present in urine for a longer time and in higher concentrations than in blood. Analysis of hair segments may define historical drug use or changes in drug habits. In Sweden the specimens of venous whole blood are taken by a nurse or physician, urine samples by the police and post‐ mortem samples (e.g. femoral blood, urine, vitreous humor, liver, brain, kidney and lung) by a forensic pathologist. After sampling all specimens are sent to one central laboratory for toxicological analysis. During the transport the samples are stored at ambient temperature for a period of about 20‐24 h. However, the blood samples contain 100 mg sodium fluoride and 25 mg potassium oxalate as preservatives and the urine samples contain 1% sodium fluoride as a preservative. Before analysis, the samples are stored in a refrigerator. The best storage temperature for most of the drugs is at 4°C for short‐term storage and at –20°C for long‐term storage [17]. For practical reasons it is most common to keep blood samples at 4°C even for long‐term storage. In Sweden the forensic laboratory has to keep blood samples in a cold place for one year to enable reanalysis if necessary.
Many substances are unstable in biological samples and undergo degradation during storage. Instability can depend on physical (e.g. type of tubes and preservatives, light, temperature), chemical (hydrolysis, oxidation) or metabolic processes (enzyme activities and/or metabolic production) [6,15,17]. In the area of analytical toxicology, the stability of drugs of abuse in biological specimens has been extensively studied (see section “Stability investigations of drugs”).
Design and evaluation of stability experiments
Stability investigations mainly comprise studies of the influence of long‐term and/or short‐term storage under the same conditions that laboratory samples are normally collected, stored and processed. But in connection with method validation also in‐process stability, freeze‐thaw stability and processed sample stability are included. Accounts and recommendations of stability experimental designs and stability evaluations are available [12,13,19], but generally accepted guidelines have not yet been established [15,20].
Several different types of stability tests including stock standard solution stability are required for complete evaluation [11‐13,19]. Long‐term stability studies usually cover a storage period that is expected for ordinary laboratory samples and under the same storage conditions used routinely. In‐process or bench‐top stability is the stability at ambient temperature over the time needed for sample preparation. During reanalysis, samples have to be frozen and thawed; therefore stability tests over multiple freeze/thaw cycles are recommended. Processed stability tests are needed to investigate stability in prepared samples e.g. sample extracts in auto sampler conditions.
Stability testing by comparing quality control samples at two concentration levels before (comparison samples/reference samples) and after (stability samples) exposing to test conditions has been suggested [12,13,19]. The reference samples can either be freshly prepared or stored below –130°C. After storage at selected temperature and time intervals in the study, reference and stability samples are analysed together and the results are compared. Stability acceptance has been recommended for concentration ratios between reference samples and stability samples of 90 and 110%, with 90%‐confidence intervals within 85‐115% [19]. The mean of the stability can also be tested against a lower acceptance limit corresponding to 90% of the mean of the reference samples using a one‐sided t test [13].
Various experimental designs and different procedures for data evaluation exist in stability investigations. Mostly, stability tests are conducted by adding (spiking) the drug (analyte) at different concentrations to a pooled drug‐free matrix (e.g. whole blood, plasma, serum and/or urine), aliquoted and stored at the same time and in the same way as ordinary samples. The concentrations are measured at selected time intervals and compared to detect any degradation trend [21‐32]. Among reported investigations, also studies on authentic material from volunteers dosed with the drug or from laboratory cases have been performed [25,32‐36].
Stability investigations have been evaluated in several different ways by statistical parametric tests like t test [26], paired t tests [32,35], analysis of variance (ANOVA) [28,36] or by nonparametric tests like Kruskal‐Wallis and Mann‐Whitney [31]. Analytes have been regarded as stable if difference between initial concentration (C0) and concentration at a given time (Ct) does
not exceeded the critical difference, d = C0 – Ct < SD of the method of analysis
[30,34]. Stability has also been evaluated on a percentage base with regard to analyte decrease or increase during storage [22,23,29,33,36].
Stability investigations of drugs
Specific stability studies on several forensically relevant drugs in human blood or tissues have been done; such as cocaine, its metabolites and its degradations
product in whole blood, post‐mortem blood or plasma [24,29], benzodiazepines, antidepressants, analgesics and/or hypnotics in whole blood, plasma or post‐
mortem blood [21,22,35,37,38], morphine and/or its glucuronides and/or
buprenorphine in whole blood, plasma or post‐mortem blood [23,25], 11‐nor‐Δ9‐
carboxy‐tetra‐hydrocannabinol glucuronide (THCCOOglu) in plasma [30], toluene and acetone in liver, brain and lungs [31], 3,4‐methylenedioxymethamphetamine (MDMA), 3,4‐methylenedioxyethylamphetamine (MDEA) and 3,4‐methylene‐ dioxyamphetamine (MDA) in whole blood [26], carbon monoxide in post‐mortem
blood [39], gamma‐hydroxybutyric acid (GHB) in blood [36] and ethanol in whole blood and plasma [32]. In urine, drug stability during storage has been investigated for drugs like 11‐nor‐Δ9‐carboxy‐tetra‐hydrocannabinol acid
(THCCOOH), amphetamine, methamphetamine, ephedrine, morphine, codeine, cocaine, benzoylecgonine and/or phencyclidine [27,33,34], lysergic acid diethylamide (LSD) [40], the LSD metabolite 2‐oxo‐3‐hydroxy lysergic acid diethylamide (O‐H‐ LSD) [28] and GHB [36].
Depending on conditions (e.g. time, temperature and/or pH) changes in drug concentrations were observed for many drugs in these experimental studies. For example, GHB in post‐mortem blood and urine (metabolic production) [36], THCCOOH in urine, THCCOOglu in urine and plasma (decarboxylation, enzymatic and chemical hydrolysis) [30,34], acetone in liver, brain and lungs (reduction) [31] and cocaine in whole blood and post‐mortem blood (enzymatic and chemical hydrolysis) [24]. In post‐mortem blood, degradation was noticed for e.g. the benzodiazepine metabolite 7‐amino‐ nitrazepam and for the hypnotic drug zopiclone [35].
Zopiclone
Zopiclone is a short‐acting hypnotic drug, a central nervous system depressant, with muscle relaxant and anticonvulsant properties. The drug was introduced for treatment of insomnia in the 1980s. In Sweden, zopiclone is a common finding in samples from drug‐impaired drivers, users of recreational drugs and forensic autopsy cases [16,41].
Zopiclone is considered a non‐benzodiazepine from the cyclopyrrolone class. It contains an asymmetric carbon atom and since all the four substituents in the molecule are different it possesses chirality. Each form (left‐ or right‐ handed) of the chiral compound, the two mirror images of the molecule, are called enantiomers or optical isomers. Zopiclone is a racemic mixture composed of the (+)‐enantiomer with S‐configuration and the (−)‐enantiomer with R‐configuration [42]. The chemical structure of zopiclone is shown below (Fig. 1). Fig. 1. Zopiclone, 6‐(5‐chloro‐ 2‐pyridyl)‐7‐(4‐methyl‐1‐ piperazinyl)carbonyloxy‐6,7‐ dihydro(5H)pyrrolo‐(3,4‐ b)pyrazin‐5‐one. Empiric formula: C17H17ClN6O3. Molecular weight: 388.81 g/mole. N N N N O N N CH3 O O Cl
* * * * * * The racemic mixture of zopiclone is sold under various brand names such as Imovane® (e.g. Sweden, Norway), Imozop® (Denmark), Zimovane® (e.g.
United Kingdom, Ireland) and Limovan® (e.g. Spain). The S(+)‐enantiomer
eszopiclone is available under the brand name Lunesta® (e.g. USA). The drugs
are used for short‐term insomnia therapy. The short‐term effects of insomnia are characterized as difficulty in falling asleep, frequent nocturnal awaking and/or early morning awaking. The usual dose of Imovane® is 5 or 7.5 mg at
bedtime [43].
Pharmacokinetics
After oral administration of the racemic mixture, zopiclone is rapidly absorbed from the gastrointestinal tract, with a bioavailability of approximately 80% [44]. Plasma protein binding of zopiclone was reported as 45% [45] in one study and 80% in another [46]. Both albumin and α 1‐acid glycoprotein contribute to protein binding but also other proteins can be involved (e.g. globulins, lipoproteins) [46]. Zopiclone is rapidly and widely distributed to body tissues including the brain, and is excreted in urine, saliva and breast milk. Zopiclone is metabolized by decarboxylation, oxidation, and demethylation. In the liver zopiclone is partly metabolized to an inactive N‐ demethylated (13‐20% of dose) and an active N‐oxide metabolite (9‐18% of dose) (Fig. 2) [44]. Fig. 2. Pathways of zopiclone metabolism in humans (*position of the asymmetric carbon). N N N N O N N CH3 O O Cl CYP3A4 CYP2C8 N N N N O N N H O O Cl CYP3A4 N N N N O N N O O Cl CH3 O Zopiclone N‐oxide‐zopiclone N‐desmethylzopiclone
The cytochrome P‐450 (CYP) enzymes CYP3A4 and CYP2C8 are involved in the metabolism of zopiclone. Both metabolites have a correlation to CYP3A4 activity but the N‐desmethylzopiclone formation also has a correlation to CYP2C8 activity [47]. Approximately 50% of the administered dose is decarboxylated to inactive metabolites of which some is excreted as carbon dioxide via the lungs. Less than 7% of the administrated dose is renally excreted as unchanged zopiclone. In urine, the N‐desmethyl and N‐oxide metabolites account for 30% of the initial dose. Elimination half‐life (t1/2) of
zopiclone is in the range of 3.5 to 6.5 hours [44].
There is no gender difference in zopiclone pharmacokinetics and patients with liver or renal dysfunction show only minor modification of the pharmacokinetic parameters, but the plasma half‐life of zopiclone increases with age [44,45].
All the pharmacokinetic processes, absorption, distribution, metabolism and excretion, are influenced by chirality. Plasma concentration of S(+)‐ zopiclone becomes higher than that of its antipode R(−)‐zopiclone after oral administration of the racemic mixture and the urine concentration of R(–)‐N‐ desmethylzopiclone and R(−)‐N‐oxide‐zopiclone become higher than that of their antipodes [48].
Drug interactions occur when the efficacy or toxicity of a medication is changed by concomitant administration of another substance. Inhibitors of CYP3A4 can increase plasma zopiclone concentrations [49,50] while CYP3A4 inducers decrease the plasma concentration of zopiclone [51].
Pharmacodynamics
GABAA receptors mediate inhibitory synaptic transmission in the central
nervous system and are the targets of neuroactive drugs used in the treatment of insomnia. GABAA receptors are pentametric membrane proteins that
operate as GABA (‐aminobutyric acid)‐gated chloride channels. Agonists increase the chloride permeability, hyperpolarize the neurons, and reduce the excitability. The receptors are made up of seven different classes of subunits with multiple variants (α1‐α6, β1‐β3, γ1‐γ3, ρ1‐ρ3, δ, ε and θ) that are differentially expressed throughout the brain. Most GABAA receptors are
composed of α‐, β‐ and γ‐subunits [52]. Zopiclone has a high affinity for the benzodiazepine binding site and acts at γ2‐, γ3‐bearing GABAA receptors,
including α1β2γ2 and α1β2γ3, but relative to benzodiazepines, produce comparable anxiolytic effects with less sedation, muscle relaxation, or addictive potential [53,54].
It has been suggested that zopiclone has a pharmacological profile different from benzodiazepines either because of a differential affinity for different GABAA receptor subtypes or partial agonistic properties. Further, it
has been found that zopiclone behaves as a partial agonist at the GABAA
receptor with a lower intrinsic activity relative to benzodiazepines. S(+)‐ zopiclone has more affinity for benzodiazepine sites than R(−)‐zopiclone, but both enantiomers are active at GABAA receptors [54‐57].
The advantage of racemic zopiclone could be seen within 30 minutes, by ease of falling asleep, increased sleep duration and decreased number of awakenings during the night [43]. Drugs that act on the central nervous system can have adverse effects on performance and behaviour of the individual. Residual effects on psychomotor and cognitive functions are dependent on dose and half‐life of the drug [58].
Forensic cases
At the clinically recommended dose of 7.5 mg, the peak plasma concentration of 0.06 mg/L was reached within 1‐2 h [43]. Blood concentrations of about 0.1 mg/L were seen following therapeutic use [59] and toxicity might occur at serum levels above 0.15 mg/L [60]. During the past few years there have been an increasing number of reports about the abuse and misuse of zopiclone. One study showed a prevalent misuse of zopiclone when its degradation product 2‐amino‐5‐chloropyridine in urine samples was detected [61]. The zopiclone distribution in cases of petty drug offences in Sweden in the 2000 to 2009 period is shown in Table 1.
Table 1. Concentrations of zopiclone in whole blood samples from cases of petty drug offences in Sweden with zopiclone detected (data collected from the laboratory database at the Department of Forensic Genetics and Forensic Toxicology). Year Number of zopiclone cases Mean value (μg/g) Median value (μg/g) Highest value (μg/g) 2000 3 0.03 0.03 0.03 2001 7 0.06 0.03 0.19 2002 12 0.18 0.16 0.50 2003 10 0.08 0.07 0.15 2004 6 0.05 0.05 0.06 2005 15 0.10 0.04 0.50 2006 22 0.12 0.06 0.90 2007 26 0.09 0.06 0.28 2008 28 0.10 0.05 0.70 2009 37 0.08 0.05 0.30
Zopiclone and other sedative hypnotic drugs are detected frequently in cases of people suspected of driving under the influence of drugs [16]. Because of the adverse effects of increased reaction time, which may affect driving performance, the drug is not suitable when skilled tasks are performed. Zopiclone concentrations in blood from drivers over a period of about six years showed that 80% had higher blood concentrations than those expected from therapeutic doses [62]. A connection between road‐traffic accidents and zopiclone use has been reported concluding that users of zopiclone should be advised not to drive [63]. The zopiclone distribution in cases of drug‐impaired drivers in Sweden in the 2000 to 2009 period is shown in Table 2.
Table 2. Concentrations of zopiclone in whole blood samples from cases of drug‐impaired drivers in Sweden (data collected from the laboratory database at the Department of Forensic Genetics and Forensic Toxicology). Year Number of zopiclone case Mean value (μg/g) Median value (μg/g) Highest value (μg/g) 2000 34 0.10 0.05 0.30 2001 59 0.09 0.07 0.44 2002 55 0.11 0.07 0.53 2003 58 0.11 0.08 0.45 2004 52 0.08 0.04 0.34 2005 59 0.12 0.09 0.41 2006 62 0.11 0.06 0.50 2007 64 0.10 0.07 0.50 2008 89 0.14 0.08 1.0 2009 108 0.10 0.08 0.40 Fatalities resulting from poisoning with zopiclone combined with alcohol or other drugs have also been described [64‐66] and cases of fatal zopiclone overdose with zopiclone concentrations of 1.4‐3.9 mg/L in the blood have been reported [67]. An overdose after ingestion of 90 mg of zopiclone has shown that this amount could be a minimum lethal zopiclone dose (in femoral blood the zopiclone concentration was 0.254 mg/L) [66]. In Sweden, zopiclone is one of the most frequently identified drugs in post‐mortem femoral blood [41]. The zopiclone distribution in forensic autopsy cases in Sweden in the 2000 to 2009 period is shown in Table 3.
Table 3. Concentrations of zopiclone in femoral blood samples from forensic autopsy cases in Sweden (data collected from the laboratory database at the Department of Forensic Genetics and Forensic Toxicology). Year Number of zopiclone cases Mean value (μg/g) Median value (μg/g) Highest value (μg/g) 2000 157 0.18 0.09 1.6 2001 165 0.29 0.09 3.1 2002 194 0.27 0.08 8.2 2003 200 0.26 0.08 5.6 2004 147 0.27 0.08 4.2 2005 226 0.34 0.08 8.6 2006 228 0.36 0.11 4.7 2007 274 0.28 0.08 13.5 2008 264 0.40 0.10 19.0 2009 277 0.27 0.09 4.7
Analytical methods
Zopiclone is a white to light yellow crystalline solid and is slightly soluble in water, soluble in most organic solvents (e.g. ethanol, acetonitrile, dichlormethane) [44]. Analysis of zopiclone in biological specimens is complicated by its instability in certain solvents as methanol, acid or basic conditions [59,68,69]. Standard solution should be prepared in acetonitrile and extraction must be done at neutral pH to ensure stability [59,70].Several analytical methods, such as high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography mass spectrometry (GC‐MS), liquid chromatography mass spectrometry (LC‐ MS) and liquid chromatography tandem mass spectrometry (LC‐MS‐MS), have been developed for the quantification of zopiclone in whole blood and plasma [69,71‐79]. Some assays (HPLC, LC‐MS, LC‐MS‐MS) have also been developed to separate and determine zopiclone and its metabolites (N‐ desmethylzopiclone and N‐oxide‐zopiclone) in urine [71,77,80,81]. One method (HPLC) has been reported to detect zopiclone and its metabolite N‐ desmethylzopiclone in plasma [78]. Stereo specific methods [LC, capillary electrophoresis (CE), radioimmunoassay (RIA) and HPLC] have been developed to separate the enantiomers [70,82‐84]. Two methods (HPLC, GC‐ MS) for detection of the zopiclone degradation product 2‐amino‐5‐ chloropyridine have been described [85,86].
Biological specimens
It has been confirmed that physical processes e.g. temperature have an effect on zopiclone stability and it has been shown that zopiclone undergoes degradation by chemical hydrolysis at basic pH by ring opening and conversion to 2‐amino‐5‐chloropyridine [68,85,86].
Specific long‐term stability investigation showed a 21% decrease of the zopiclone concentration in post‐mortem femoral blood after storage for twelve months at –20°C [35].
Data on zopiclone stability have also been carried out from stability experiments in connection with method development and validation. Long‐
term stability tests have shown that zopiclone in spiked human plasma is stable
for one month [69,78] and for six months [70] when stored at –20°C or lower.
Freeze‐thaw stability tests have indicated zopiclone stability in spiked human
plasma for three freeze‐thaw cycles [69,78]. Short‐term or in process stability tests have shown no evidence of degradation in plasma quality control samples stored at room temperature for 24 h [78]. No loss of zopiclone could be detected following storage of blood samples (plasma) at 4‐8°C for less than 6 h, but the concentration of zopiclone in blood was reduced by 25% and 29% for the (–)‐ and (+)‐enantiomers, respectively, after 20 h at ambient temperature [70]. Processed sample stability or sample extract reanalysis has shown that zopiclone is stable for up to 24 h in water‐methanol extract [78] but unstable in ethanol extracts [69].
Zopiclone in whole blood is analyzed in forensic toxicology and a detailed stability investigation of zopiclone in this matrix is necessary. Both albumin and α‐1‐acid glycoprotein are involved in zopiclone protein binding [46]. Protein binding might protect drugs from in vitro breakdown, but no relationship between concentration decrease during storage and drug binding has been confirmed [21]. However, considering individual differences in plasma protein concentrations or possible plasma protein binding competition between different drugs in vitro, stability studies on authentic samples might be important.
AIMS OF THESIS
The overall aim of this thesis was to investigate the stability of zopiclone in human whole blood and to study stability differences between authentic and spiked samples. Depending on handling, storage and specimen conditions, the zopiclone concentration is likely to change. Since interpretation of zopiclone concentrations in whole blood are important in forensic toxicology, detailed knowledge of the zopiclone stability in whole blood matrices is essential for reliable analysis and interpretation of results.
Specific aims
Paper I
To investigate the stability of zopiclone in human blood during storage under different conditions, stability differences between authentic and spiked blood, freeze‐thaw and processed sample stability.Paper II
To compare stability between authentic and spiked blood samples from the same donor and, in particular, to investigate the influence of short‐term pre‐ analytical storage conditions.
MATERIALS AND METHODS
Study designs
Long- and short-term stability
Pooled human drug‐free whole blood was used as matrix for spiked samples and this was obtained from the local blood bank at the University Hospital in Linköping. To 1 g blood, zopiclone was added from stock standard solutions to give target concentrations of 0.2 μg/g (low level) and 0.5 μg/g (high level), respectively. These levels were chosen on the basis of concentrations found in authentic blood samples and being high enough to follow decrease over time. After the initial measurement of five replicates at each level, the remaining tubes were stored at –20°C, 5°C or 20°C. The initially measured mean concentration was used as starting value. To investigate the effect of time and temperature, the zopiclone concentration was measured in duplicate at selected times during twelve months.
Authentic and spiked blood was used to investigate stability differences under the conditions usually encountered in our laboratory. Authentic blood samples from nine cases were included, where venous blood specimens had been obtained by medical personnel and sent by the police to the Department of Forensic Genetics and Forensic Toxicology, Linköping, for analysis. Aliquots were reanalyzed after 1, 3, 5 and 8 months of storage at 5°C and the concentration of zopiclone was measured. The first measured concentration was used as starting value. Spiked blood samples were prepared to compare with the authentic samples. To 1 g aliquots of drug‐free blood, zopiclone standard solution was added at ten different concentrations (0.02, 0.03, 0.06, 0.09, 0.10, 0.15, 0.20, 0.30, 0.40 and 0.50 μg/g) and after initial measurement the spiked samples were stored at 5°C. The initially measured concentration was used as starting value. The zopiclone concentration was measured in duplicate after 1, 3, 5 and 8 months of storage.
Freeze-thaw stability
Aliquots of drug‐free blood were spiked in triplicates with zopiclone standard solution to one low concentration (0.02 μg/g) and one high concentration (0.2 μg/g). The samples were analyzed before and after three freeze (20 ± 2 h at –20˚C) ‐ thaw cycles (in total less than 2 h at room temperature). Also, authentic blood samples were used. Aliquots from eight samples were analyzed, then frozen at −20°C for 20 ± 2 h, thawed at room temperature for less than 2 h, and reanalyzed. Because of limited authentic material, the authentic samples could only go through one freeze‐thaw cycle.
Processed stability
Six samples were reinjected to evaluate processed sample stability of zopiclone concentrations in extracts of butyl‐acetate. The extracts were reinjected 21 h (one day) and 45 h (two days) after on‐instrument storage at ambient temperature.
Degradation
In addition to the stability investigation, the formation of zopiclone degradation products was investigated in blood samples during storage for 24 h at 37°C. To 1 gram of drug‐free blood 0.3 μg/g of zopiclone (0.77 nM) was added and the aliquots were incubated 0, 1, 3, 6, 18, and 24 h before extraction and analysis.
Influence of pre-analytical conditions
Nine healthy drug‐free volunteers participated in the study approved by the regional ethics committee in Linköping, Sweden (#M164‐08). Before oral administration of a single dose of 10 mg Imovane®, blood samples were drawn
in tubes containing EDTA (2 x 3 mL) and tubes containing sodium fluoride and potassium oxalate (6 x 9 mL). After administration, at the estimated peak time of 1.5 h, blood samples were taken in tubes containing sodium fluoride and potassium oxalate (6 x 9 mL).
The EDTA blood samples were directly transported to a local clinical laboratory (at University Hospital, Linköping) for analysis of erythrocyte volume fraction, plasma albumin and plasma α‐1‐glycoprotein. The pre‐dosed blood was pooled individually; then divided into glass tubes and spiked with stock standard solution of zopiclone to give target concentrations of 0.15 μg/g (n = 4) and 0.08 μg/g (n = 5), respectively. The levels were expected to reflect authentic blood levels. The post‐dosed blood was pooled individually and aliquoted in glass tubes.
After the initial concentration was measured in triplicate, the remaining tubes were stored at 20°C, 5°C or –20°C. The initially measured mean concentration was used as starting value. Measuring started within 8 ± 1 h after the first sampling. To investigate the effect of time and temperature in authentic and spiked blood, the zopiclone concentration was measured in triplicate at selected times after storage (days at 20°C, weeks at 5°C and months at –20°C).
Ethical considerations
In the first study (Paper I), biological material from forensic cases was used. Data was collected from a laboratory database at the Department of Forensic Genetics and Forensic Toxicology. The samples were reanalyzed and evaluated unidentified with no connection to the cases. In the second study (Paper II), biological material from volunteers was used. Research procedures adhered to obligations to the study participants and the protocol was approved by the Regional Ethics committee, Faculty of Health Sciences, Linköping University, Sweden (# M164‐08).
Equipment
Vacutainer tubes and/or VenoSafe tubes (Terumo Europe NV, Leuven, Belgium) containing 100 mg sodium fluoride and 25 mg potassium oxalate as preservatives and Vacutainer tubes (BD Vacutainer, Plymouth, United Kingdom) containing EDTA were used for blood sample collection. Aliquots of 1 g of whole blood were stored in glass tubes (DURAN®, Mainz, Germany).
Eppendorf pipettes (accuracy and precision controlled each four months) were used. Blood samples were weighed on a Sartorius LC421 scale (calibrated once
a year and controlled in house each month). Hettich (Universal 30 RF) centrifuge was used for centrifugation (calibrated once a year).
Chemicals and solutions
Acetonitrile, methanol, n‐butyl acetate, methyl‐tertbutylether of analytical grade came from Merck (Darmstadt, Germany). Hydrochloric acid, formic acid, sodium hydroxide, tris(hydroxymethyl)aminomethane, potassium dihydrogen phosphate and dipotassium hydrogen phosphate * 3 H2O were
supplied by Merck (Darmstadt, Germany) and ammonium formiate by Sigma Aldrich (Steinheim, Germany). The 0.5 M phosphate buffer pH 7.0 used in extraction was prepared by mixing a 0.5 M dipotassium hydrogen phosphate solution with 0.5 M potassium dihydrogen phosphate solution to pH 7.0. The 1 M trisbuffer was adjusted to pH 11. Reference solutions (stock standard solutions) were prepared in acetonitrile with certified zopiclone (Sigma‐ Aldrich, St. Louis, MI, USA) to concentrations of 0.1 mg/mL, 0.01 mg/mL, 1 μg/mL and 0.1 μg/mL. The reference solutions were stored at ‐20°C with a tested stability of at least 12 months. Prazepam (Sigma‐Aldrich, St. Louis, MI, USA) was prepared in methanol and used as internal standard (IS) with a concentration of 0.01 mg/mL. The breakdown product 2‐amino‐5‐ chloropyridine was obtained from Sigma‐Aldrich (Steinheim, Germany). Deionized water was prepared on a Direct Q laboratory plant (Millipore).
Analytical methods
Gas chromatography
The measurement principle GC‐NPD was used for zopiclone quantification. To 1.0 g of the sample 30 L internal standard (prazepam 0.01 mg/mL) and 0.5 mL 0.5 M phosphate buffer pH 7.0 were added. Extraction was made with 0.3 mL butyl‐acetate for 10 minutes and after phase separation by centrifugation (5000 rpm), the organic extract was transferred to a sampler vial and analyzed by GC‐NPD.GC‐NPD analyses were performed on a Hewlett Packard (HP) 5890 GC (Waldbronn, Germany). 5 μL aliquots of the extract were injected with
automatic injector HP 7634A at 250°C into an analytical column, DB‐5 capillary column 15 m by 0.25 mm ID and 0.25 m thickness (J&W, Folsom, CA, USA). The initial oven temperature was 200°C and was then programmed to 300°C at a rate of 25°C /min, held at 300°C for 4 min before an increase to 320°C. The carrier gas was helium with a constant pressure. The detector temperature was 320°C. Results were evaluated using Chromeleon version 6.7 (Dionex Corparation, Sunnyvale, CA, USA). Example of a typical chromatogram is shown in Fig. 3.
Fig. 3. Example of chromatogram from blood extract of a control sample at low level 0.02 μg/g. Internal standard is prazepam (4.89) and the peak before zopiclone (7.48) is cholesterol.
This GC‐NPD assay is routinely used and method validation was performed before the stability investigations. Calibration curves were made by adding zopiclone reference solution at five different concentrations (0.01, 0.05, 0.2, 0.5 and 1 μg/g) to drug‐free fresh whole blood and analyzed in duplicate. Linear calibration curves were obtained in the range of 0.01 to 2 μg/g. Precision was expressed as the percentage coefficient of variation (%CV) of measurements. Inter‐ and intraday variability were determined at two levels (0.02 and 0.50 μg/g). Intra‐day variability (n = 5) was 6.1%CV and 4.4%CV and inter‐day variability (n = 45) was 26.6%CV and 24.4%CV for the low and high level, respectively. The limit of detection (LOD) was 0.007 μg/g and the limit of 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 mV min 4.89 7.48 5 10 15 20 25 Zopiclone Internal Standard 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 mV min 4.89 7.48 5 10 15 20 25 Zopiclone Internal Standard 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 mV min 4.89 7.48 5 10 15 20 25 Zopiclone Internal Standard
quantification (LOQ) was 0.02 μg/g. Recovery in the assay was 51%. Zopiclone metabolites and degradation products were not included in this method.
Liquid chromatography
To identify and quantify zopiclone breakdown products a brief experiment was performed. After the incubation at 37°C, 0.5 mL 1 M trisbuffer was added to the samples. Extraction was made with methyl‐tertbutylether for 10 minutes and after phase separation by centrifugation (5000 rpm), the organic extract was removed and evaporated with nitrogen. The residue was reconstituted in 0.2 mL of a 50% solvent mixture of A and B (see below) and analyzed by LC‐ MS‐MS.
The LC/MS/MS system consisted of a Waters ACQUITY UPLC® (Ultra
Performance LC) with a Binary Solvent Manager, Sample Manager, and Column Manager (Waters Co., Milford, MA, USA) connected to an API 4000™ triple quadrupole instrument (Applied Biosystems/MDS Sciex, Stockholm, Sweden) equipped with an electrospray interface (TURBO V™ source, TurboIonSpray® probe) operating in the MRM mode.
The acquisition method included two transitions for zopiclone (389.1/217.0 and 389.1/245.1), N‐desmethyl‐zopiclone (375.1/217.1 and 375.1/245.0), N‐ oxide‐zopiclone (405.2/143.1 and 405.2/245.0), and 2‐amino‐5‐chloropyridine (128.9/112.1 and 128.9/76.1). Ultra Performance Liquid Chromatography (UPLC®) was performed using a 1.7 μm, 50 × 2.1 mm ACQUITY UPLC®
ethylene bridged hybrid (BEH) C18 column (Waters Co.), operated in gradient
mode at 0.6 mL/min with a total run time of 3 min. Solvent A consisted of 0.05% formic acid in 10 mM ammonium formiate and Solvent B of 0.05% formic acid in acetonitrile. External calibration curves were used for quantitation.
Quality controls
Quality controls at two levels, low (0.02 μg/g) and high (0.50 μg/g) were extracted and measured together with each batch of samples in the long‐ and short‐term stability investigation (GC‐NPD). Quality control samples at three levels, negative, low (0.02 μg/g) and high (0.20 μg/g), were used in the same way in the freeze‐thaw and in the processed stability investigations as well as in the study of the influence of pre‐analytical conditions. The quality control samples were freshly prepared before each assay by spiking drug‐free blood
matrix with the reference standard solution at each concentration. Method precision (random error, variation) and accuracy (systematic error, mean bias) were evaluated for these quality control samples.
Clinical chemical analysis
The erythrocyte volume fraction, plasma albumin and plasma α‐1‐ glycoprotein were analyzed at the clinical laboratory at University Hospital, Linköping. Erythrocyte volume fraction (EVF) was measured by impedance technique (Cell‐Dyn Sapphire, Abbot Laboratories, Saint‐Laurent, Québec, Canada), plasma albumin and plasma α‐1‐glycoprotein were analyzed by immuno chemical technique (ADVIA 1800, Siemens, Deerfield, IL, USA and BN ProSpec, Siemens, Deerfield, IL, USA).
Statistical analysis
The descriptive statistics used were means and standard deviation. Wilcoxon Signed Ranks Test was used, when comparison between the different storage times was evaluated. When differences between authentic and spiked samples were evaluated, Mann‐Whitney U‐test was used. A probability of less than 5% (p < 0.05) was regarded as significant. The statistical analysis was performed using SPSS 16.0 for Windows in Paper I and SPSS Statistics 17.0 in Paper II. In paper II the stability was also evaluated by using a lower acceptance limit (initial value – 2 x SD of method). The ± 2 standard deviations (SD) are a common limit for accepting batch controls in a series of samples with a 95% confidence.
RESULTS
Paper I
Long-term and short-term stability
Effect of time and temperature
Long‐ and short‐term stability was investigated at two concentrations of zopiclone and at three different storage conditions. A significant difference between samples stored at –20°C, 5°C or 20°C was found (p < 0.05). The results from the higher concentration (0.59 μg/g) and the lower concentration (0.23 μg/g) showed the same degradation profile with no significant differences. Data from samples at the high concentration (0.59 μg/g) are depicted in Fig. 4 showing that zopiclone is most stable at –20°C. After five months 74‐88% of the initial zopiclone concentration was left of the low and high levels, respectively. However, after six months of storage some decrease in zopiclone concentration was noticed which reached a 36‐52% decrease in the end of studied period. In samples stored at 5°C degradation of zopiclone was seen already after a few weeks. After four weeks, the residue was 42‐61%, after three months 12‐13%, and after five months there was no measurable concentration left. At a storage temperature of 20°C, the concentration decreased more than 50% after the first three days of storage.
0 50 100 150 0 50 100 150 200 250 300 350 400 Time (days) Re ma ining c onte n t (%) -20ºC 5ºC 20ºC Fig. 4. Effect of time and storage conditions in whole blood containing 0.59 μg/g of zopiclone (high level).
Authentic and spiked stability samples
As regards long‐term stability, authentic and spiked samples were compared during storage at 5°C. Zopiclone degradation over time was seen in both authentic and spiked blood samples stored at 5°C (Fig. 5). 0 20 40 60 80 100 120 140 0 50 100 150 200 250 Time (days) Remaining content (%) Authentic Spiked Expon. (Authentic) Expon. (Spiked) Fig. 5. Authentic and spiked samples (at levels between 0.01‐0.49 μg/g) during eight months of storage at 5°C. Within the authentic group, storage before first measurement varied between 2‐14 days. For all the spiked samples first measurement was at day zero.
A significant difference between the initial content and the residue was noticed already after one month (p < 0.05) in the spiked group as well as in the authentic group (Table 4). Mann‐Whitney U‐test showed no significant differences between the two groups, but within both of the groups the remaining content varied. Additionally, in the authentic group the first measured concentration was used as starting value while the initially measured concentration was used as starting value in the spiked group (Fig. 5).
Table 4. A compilation of zopiclone remaining content in authentic and spiked human blood samples during storage for 8 months at 5°C. Mean remaining content (%) and (range) after storage in months Material Initial content (%) 1 3 5 8 Authentic (n = 9)* 100 75** (50‐133) 13** (0‐38) 6** (0‐23) 0** 0 Spiked (n = 10) 100 77** (55‐100) 19** (0‐50) 8** (0‐25) 0.9** (0‐4)
*Other drugs, e.g. alprazolam, carisoprodol, citalopram, codeine, diazepam, dihydropropiomazine, ephedrine, ethanol, lamotrigine, levomepromazine, meprobamate, nordazepam, oxazepam and/or tramadol were present in eight of the nine authentic samples. Within the authentic group, storage before first measurement varied between 2‐14 days.
**p < 0.05
Freeze-thaw stability
Freeze‐thaw stability of zopiclone in whole blood was evaluated in both authentic and spiked samples. There were no differences in zopiclone concentration before and after the freeze‐thaw cycle for any of the eight authentic samples with a zopiclone concentration ranging from 0.03 to 0.15 μg/g. In the spiked samples, there were no differences at the low level (0.02 μg/g) after three freeze‐thaw cycles, but some decrease (8%) at the high level (0.20 μg/g) was noticed after three cycles.
Processed stability
After storage on the analytical instrument during two days at ambient temperature, no evidence of degradation was seen in the processed sample stability test of the sample extract reanalysis.
Degradation products
The zopiclone degradation was investigated in spiked samples during 24 h incubation at 37°C. In these samples the zopiclone metabolites N‐desmethyl‐ zopiclone and N‐oxide‐zopiclone were not detected. However, 2‐amino‐5‐ chloropyridine was formed in approximately equimolar amounts to zopiclone decay (Fig. 6). 0,0 0,2 0,4 0,6 0,8 1,0 0 5 10 15 20 25 Time (h) C oncentration (nM) Zopiclone ACP Fig. 6. Degradation of zopiclone and formation of 2‐amino‐5‐chloropyridine (ACP) in spiked whole blood at 37°C.
Quality control samples
Results from the quality control samples during short‐ and long‐term stability investigation in Paper I are shown in Table 5. H N Cl 2N N N N N O N N CH3 O O Cl
Table 5. The descriptive statistics for the quality controls in the long‐ and short‐term zopiclone stability investigation: a) spiked blood (during 12 months) and b) authentic and spiked blood (during 8 months). L = low level and H = high level. Quality Control Theoretical value (μg/g) Mean value (μg/g) SEM SD Precision CV% Mean Bias Accuracy % a) L (n = 75) 0.02 0.014 0.0005 0.004 27 ‐0.006 70 a) H (n = 75) 0.50 0.444 0.011 0.092 21 ‐0.056 89 b) L (n = 48) 0.02 0.015 0.0004 0.003 20 ‐0.005 75 b) H (n = 48) 0.05 0.440 0.011 0.076 17 ‐0.060 88
Paper II
Authentic and spiked stability samples
Stability differences between authentic and spiked blood samples from the same donor were compared.
The spiked levels were intended to reflect those in the authentic samples. However, the dosed subjects presented with zopiclone blood levels lower than expected and therefore four of the spiked samples were excluded (spiked at 0.15 μg/g). Zopiclone concentrations in the authentic samples 90 minutes after oral administration varied between 0.017–0.060 μg/g (n = 9) and the target concentration in the spiked samples was 0.08 μg/g (n = 5). The measured concentration at the time of spiking ranged between 0.064‐0.083 μg/g. The remaining content of zopiclone in authentic and spiked blood showed the same decreasing trends at 20°C and 5°C as depicted in Fig. 7.
